Promoter regions are essential for driving and regulating gene expression. These stretches of DNA sequences are usually found upstream of the gene open reading frame. The key genetic elements in a promoter include enhancer sites, silencing sites, upstream activator sequences, transcription factor binding sites and RNA polymerase binding sites (e.g. Lewin, B., Genes V, Oxford University Press, 1994). These elements respond to the external environment of a given organism therefore regulating gene expression according to the growth conditions.
Strong promoters are required for efficient gene expression. Among the best promoters described for filamentous fungi so far include the Trichoderma reesei cellobiohydrolase 1 (cbh1) promoter (Ilmén, M. et al., 1996. Mol. Gen. Genet. 251, 451-460) and Aspergillus niger var. awamori glucoamylase A promoter (Ward, M. et al., 1990. Bio/Technology 8:435-440). However, both these promoters are inducible by an appropriate carbon source (eg. cellulose and starch respectively) and repressed by glucose (ie. regulated by catabolite repression). So far, natural constitutive or carbon catabolite repression insensitive promoters comparable in strength to the cbh1 and glaA have not been described.
Strategies described for promoter isolation include (i) the use of promoter probe vectors, where genomic DNA fragments are randomly cloned in front of a reporter gene which is expressed only when the cloned fragment contains promoter activity (e.g. Neve et al., 1979. Nature 277:324-325); (ii) isolation of genes and their promoters from gene libraries using hybridization based on gene specific probes (e.g. Vanhanen, S., et al., 1989. Curr. Genet. 15:181-186) or nucleic acid sequences deduced from amino acid sequences; (iii) differential hybridization of a gene bank with an induced and non-induced cDNA probe (e.g. Teeri et al., 1983. Bio/technology 1:696-699).
The present invention uses a new strategy termed proteome display for gene promoter isolation. This is different to the nucleic acid based strategies such as those listed above. Proteomic analysis typically involves separation of proteins in a sample by their isoelectric point and molecular mass (2-dimensional gel electrophoresis, 2-DE). The gels are then stained to visualize the protein spots and may be blotted onto a suitable membrane for further processing. The 2-DE technology enables separation of several hundreds of proteins in complex mixtures (O'Farrell, P. H., et al., 1975. J. Biol. Chem. 250:4007-4021) and detection of proteins that are highly expressed in an organism grown under given cultivation conditions (e.g. according to the carbon source). Protein spots in a proteomic display differ in their intensity reflecting the level they are expressed. Therefore, detection of a strongly expressed protein indicates a presence of a strong promoter driving the gene encoding that particular protein. Strongly expressed protein spots can be cut out and analyzed by mass spectrometry (Verrils, N. M., et al., 2000. Electrophoresis 21:3810-3822). The obtained amino acid sequence can be used to design oligonucleotide primers for Chromosome Walking PCR (Morris, D., et al., 1995. Appl. Environ. Microbiol. 61:2262-2269) in order to isolate the promoter from a particularly strongly expressed gene (protein). Protein identification involves comparison of the obtained peptides and/or amino acid sequences to databases available e.g. on the WWW. However, identification of a strongly expressed protein is not necessary for the isolation of the promoter driving its expression.
The hex1 gene encodes a hexagonal (HEX1) protein of the fungal Woronin body and is unique to filamentous fungi (Tenney, K., et al., 2000. Fungal. Genet. Biol. 31:205-217). The HEX1 protein was identified as a dominant protein in cell wall extracts prepared from Trichoderma reesei cultures grown on cellobiose-lactose-soybean extract medium or glucose as a carbon source, by 2-dimensional gel electrophoresis (Lim et al., 2001. Proteomics 1:899-910). Therefore, identification and isolation of the hex1 gene promoter based on a proteomic display provides a new strategy for promoter isolation and gene expression using a promoter not necessarily affected by carbon catabolite repression.
The present inventors have now devised methods for obtaining or detecting new promoters of genes.